U.S. Pat. No. 4,546,082 describes the .alpha.-factor precursor gene of Saccharomyces cerevisiae and purports to show DNA constructs of the S. cerevisiae leader sequence and heterologous genes that are useful for the secretory expression of the heterologous genes. EPA 0 116 201 describes the actual use of the S. cerevisiae .alpha.-factor leader sequence to obtain secretory expression of human epidermal growth factor in yeast. Copending U.S. patent application Ser. No. 522,909, filed 12 August 1983, which is, in part, commonly owned with the present application, describes the use of "spacer-less" S. cerevisiae .alpha.-factor leader sequences to obtain more efficient secretory expression of heterologous genes.
Science (1985) 229;1219-1224 reports the use of S. cerevisiae .alpha.-factor sequences to direct the secretion of prochymosin. It was reported that most of the prochymosin produced was present in an intracellular insoluble form rather than being secreted. Although the invertase leader was more effective at directing secretion of activatable prochymosin, an extensive mutant screen was necessary to yield reasonable amounts of prochymosin secretion.
There has been no previous demonstration of the production of mating pheromones (.alpha.-factor and a-factor) by Kluyveromyces lactis cells or other Kluyveromyces species. This failure to identify such peptides in K. lactis may, in part, be due to the fact that mating in K. lactis is inducible; whereas in S. cerevisiae the mating pheromones are produced constitutively.
Notwithstanding the availability of the mentioned leader sequences for directing secretion of heterologous polypeptides in yeast, there continues to be a need for alternative sequences that may afford more efficient or more practical production of particular polypeptides. Accordingly, applicants sought to determine whether a K. lactis .alpha.-factor exists and, if so, whether it might be useful for directing secretion of heterologous polypeptides, such as prochymosin, in yeast.